Process for the Preparation of Amides

ABSTRACT

A novel biotechnological process for the preparation of nitriles, starting from amides, is described. Micro-organisms of the genus  Amycolatopsis, Actinomadura  or  Rhodococcus  are employed for this process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 11/495,035, filed Jul. 28, 2006, which is a divisional of U.S. patent application Ser. No. 10/234,088, filed Sep. 3, 2002 (incorporated by reference in its entirety herein), now U.S. Pat. No. 7,105,322, which is a divisional of Ser. No. 09/463,203, filed Mar. 27, 2000, now U.S. Pat. No. 6,444,451, which is a national phase application under 35 U.S.C. §371 of International Application No. PCT/EP98/04587, incorporated by reference herein, which was filed on Jul. 22, 1998 and published in German on Feb. 4, 1999.

FIELD OF THE INVENTION

The invention relates to novel microorganisms of the genus Actinomadura, Amycolatopsis or Rhodococcus, and to a novel process for the preparation of amides using these microorganisms or using enzyme extracts of these microorganisms.

BACKGROUND

For amides such as, for example, nicotinamide, a vitamin of the vitamin B complex which is essential to animals and man, a number of biotechnological processes are already known. Generally, it is known that microorganisms containing nitrile hydratase convert nitriles to the corresponding amides. Thus EP-A-0 188 316 describes a process for the preparation of nicotinamide starting from 3-cyanopyridine using microorganisms of the genus Rhodococcus, Arthrobacter or Microbacterium.

A disadvantage of this process is that these micro-organisms have only a low activity for the conversion of 3-cyanopyridine to nicotinamide.

EP-A-0 307 926 describes the conversion of 3-cyano-pyridine to nicotinamide by means of microorganisms of the species Rhodococcus rhodochrous J1. In order that these microorganisms catalyse the desired conversion, they must be induced.

A further disadvantage of this process is that Rhodococcus rhodochrous J1 is red-coloured and accordingly a discoloration of the product takes place. In addition, this microorganism has a low heat stability and is inhibited, for example, by the substrate 3-cyano-pyridine.

A further process for the preparation of nicotinamide starting from 3-cyanopyridine by means of microorganisms of the species Rhodococcus rhodochrous J1 is described in EP-A-0 362 829. In order to increase the specific activity of the microorganisms containing nitrile hydratase, urea or a urea derivative was added to the culturing medium as an inducer. As in the process described beforehand, a discoloration of the product also takes place in this process.

In addition, WO 95/17 505 describes a process for the preparation of aromatic amides starting from the corresponding nitriles by means of microorganisms of the species Rhodococcus rhodochrous M33. A disadvantage of this process is the red colouration of Rhodococcus rhodochrous M33 and also the high K_(M) value for the substrate 3-cyanopyridine.

SUMMARY OF THE INVENTION

The object of the present invention was to eliminate these disadvantages and to make available a process for the preparation of amides in which the corresponding amides can be isolated in good yield and purity.

This object is achieved by the novel microorganisms according to claims 1 and 3, and by the process according to claim 6.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the determination of the molecular weight of nitrile hydratase by chromatography on TSK gel G3000 SW. The molecular weight of nitrile hydratase is shown compared to other known enzymes.

FIG. 2 shows the determination of the molecular weight of the α and β subunits of nitrile hydratase by means of SDS-PAGE. The molecular weight of the submits is show compared to other known enzymes.

FIG. 3A-B shows the absorption spectrum of the purified nitrile hydratase enzyme. A broad absorption of 300-400 nm was observed. The absorption spectrum was determined for nitrile hydratase at (A) 7.5 mg/ml and (B) 0.752 mg/ml.

FIG. 4 shows the biotranformation activity of NA40 cells under standard reaction conditions after a 30 min incubation at different temperatures, relative to activity under standard conditions. Cells were incubated at temperatures of 0° C., 10° C., 20° C., 30° C., 40° C., 50° C., 60° C., 70° C., and 80° C. for 30 min, followed by biotranformation activity testing under standard reaction conditions.

FIG. 5 shows the biotranformation activity of NA40 cells at different reaction temperatures relative to the standard reaction temperature of 20° C. Reactions were carried out in standard reaction mixture for 10 minutes at temperatures of 10° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., and 60° C.

FIG. 6 shows the biotranformation activity of NA40 cells in different 0.1M buffers relative to the standard potassium phosphate buffer. Reactions were carried out in standard reaction mixture for 10 minutes in which the potassium phosphate buffer had been replaced with glycine/HCl, citric acid/sodium citrate buffer, Tris/HCl buffer, or glycine/NaOH buffer.

FIG. 7 shows the biotranformation activity of NA40 cells at pH 7.0 after a 30 min incubation at different pH levels, relative to activity under standard conditions. Cells were incubated for 30 min at 20° C. in 42.5 mM of glycine/HCl buffer, citric acid/sodium citrate buffer, Tris/HCl buffer, or glycine/NaOH buffer, followed by biotranformation activity testing in 0.1M phosphate buffer pH 7.0 supplemented with 3-cyano-pyridine for 10 min under standard conditions.

FIG. 8 shows the biotranformation activity of nitrile hydratase at different pH levels. The conversion of 3-cyanopyridine to nicotinamide was carried out at 20° C. in a 2.0 ml reaction mixture comprising 42.5 mM citric acid/sodium citrate buffer, potassium phosphate buffer, Tris/HCl buffer, or H₃BO₃/NaOH buffer; 1.71 units of purified enzyme; and 500 mM 3-cyanopyridine.

FIG. 9 shows the biotranformation activity of nitrile hydratase under standard conditions following a 1.0 h incubation in buffers of different pH levels, relative to activity under standard conditions. 4.2 units of purified enzyme were incubated at 20° C. for 1 h in 45 mM of citric acid/sodium citrate buffer, potassium phosphate buffer, Tris/HCl buffer, or H₃BO₃/NaOH buffer. 1.71 units of the incubated solution were added to the standard reaction mixture, and the biotranformation activity of the enzyme was determined.

DETAILED DESCRIPTION

According to the invention, the process is carried out by converting a nitrile, as substrate, to the corresponding amide by means of microorganisms of the genus Actinomadura, Amycolatopsis or Rhodococcus, using an enzyme extract of these microorganisms or by means of purified nitrile hydratase of microorganisms of the genus Amycolatopsis or Actinomadura.

The nitriles employed for the biotransformation such as, for example, 3-cyanopyridine are commercially available compounds.

The microorganisms according to the invention are able to convert nitriles as substrates into the corresponding amides. Preferably, these microorganisms have the ability to grow on nitriles or amides as the sole C and/or N source.

The microorganisms according to the invention are obtainable by means of suitable selection, for example, from soil samples, sludge or waste water with the aid of customary microbiological techniques. Expediently, the microorganisms are selected by growth with nitriles or amides as the preferably sole C and N source in the presence of cobalt ions. Nitriles and amides suitable for selection are, in particular, the nitriles also employed as substrates in the later biotransformation and the corresponding amides obtainable therefrom. Suitable growth media are likewise known to the person skilled in the art, for example the medium described in Table 1 can be used.

Customarily, the microorganisms are cultured (grown) in the same manner even before the actual biotransformation, the abovementioned media being used.

As known professionally, a nitrile hydratase is only formed when the growth medium contains cobalt ions as a cofactor. Suitable “cobalt compounds generating cobalt ions” are Co²⁺ or Co³⁺ salts. Examples of Co²⁺ and Co³⁺ salts are cobalt chlorides, cobalt sulphates and cobalt acetates.

Expediently, the cobalt compound employed is a Co²⁺ salt such as, for example, CoCl₂. Growth, however, can also be carried out in the presence of vitamin B12 together with metallic cobalt or other cobalt compounds which generate a cobalt ion in situ. Expediently, the cobalt compound is employed in an amount from 1 to 10 mg/l, preferably from 1 to 3 mg/l.

Customarily, growth is carried out at a temperature from 20 to 50° C. and at a pH between pH 5 and pH 8, preferably from 30 to 45° C. and between pH 5.5 and pH 7.5.

The actual biotransformation can be carried out using microorganisms of the genus Actinomadura, Amycolatopsis, using an enzyme extract of these microorganisms or by means of purified nitrile hydratase from these microorganisms. Expediently, the biotransformation is carried out using microorganisms of the species Actinomadura spadix, for example the isolates Actinomadura spadix E3733, Actinomadura spadix E3736, Actinomadura spadix 45A32, Actinomadura spadix 4501 or Actinomadura spadix C15. The biotransformation is preferably carried out using microorganisms corresponding to the species Amycolatopsis NE 31 and Amycolatopsis NA40 or their functionally equivalent variants and mutants. Micro-organisms corresponding to the species Amycolatopsis NA40 are particularly preferably employed. Microorganisms of the species mentioned were deposited on 3 Jun. 1997 in the Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures GmbH], Mascheroderweg 1b, D-38124 Brunswick under the designations Amycolatopsis NE 31 and Amycolatopsis NA40 according to the Budapest Convention and have the deposit numbers DSMZ 11616 and DSMZ 11617 respectively. These two microorganisms have been more accurately identified and are to be assigned to species of the genus Amycolatopsis which have not yet been described in the literature.

Accordingly, the invention also relates to microorganisms of the genus Amycolatopsis or Actinomadura which are capable of converting an amide into a nitrile, in particular microorganisms with the designation Amycolatopsis NA40 (DSMZ 11617) and Amycolatopsis NE31 (DSMZ 11616).

In addition, it has been found that specific microorganisms of the genus Rhodoccus have better properties for the conversion of nitriles to amides than the Rhodococcus rhodochrous J1 described in EP-A-0 362 829. These microorganisms are Rhodoccus GF674, Rhodoccus GF578. Rhodoccus GF473, Rhodoccus GF270 (DSMZ 12211) and Rhodoccus GF376 (DSMZ 12175) or their functionally equivalent variants and mutants. The microorganism DSMZ 12175 was deposited on 15 May 1998 and the microorganism DSMZ 12211 on 8 Jun. 1998 in the Deutschen Sannlung von Mikroorganismen und Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures GmbH] according to the Budapest Convention.

The Rhodoccus strains GF270, GF376, GF473, GF578 and GF674 have been assigned according to identification to species of the genus Rhodoccus which are not yet described in the literature. Accordingly, the invention also relates to the microorganisms Rhodoccus GF270, Rhodoccus GF376, Rhodoccus GF473, Rhodoccus GF578 and Rhodoccus GF674.

Unlike the microorganisms of the genus Actinomadura or Amycolatopsis, the microorganisms of the genus Rhodoccus are expediently induced before the actual conversion. Suitable inducers are those described in EP-A-0 307 926, such as, for example, acetamide, butyramide, methacrylamide, propionamide, crotonamide and valeramide.

“Functionally equivalent variants and mutants” is understood as meaning microorganisms which are derived from the abovementioned source organisms and essentially have the same characteristics and functions as these. Variants and mutants of this type can be formed by chance, e.g. by UV irradiation or by mutagenic chemicals. Identification of Amycolatopsis NA40 Colour of aerial mycelium white Colour of substrate mycelium orange Colour of diffused pigment — Sugar spectrum ARA + GAL + MAD − XYL − GLU tr RIB + Type A DAP DL Menaquinones (in %) 8/4 − 9/0 (+) 9/2 + 9/4 +++ 9/6 − 9/8 − 16S rDNA homology 96.9% Phospholipids such as not investigated PE, OH-PE, lyso PE, met PE, PC, NPG, PI, PIM, PG, DPG, GL Fatty acids iso 16 +++ iso 15 + iso 17 (+) anteiso 15 (+) anteiso 17 (+) 10-Me 16 − 10-Me 17 + 2-OH 15 + 2-OH 16 + Type 3f MS — Identification of Amycolatopsis NE31 Colour of aerial mycelium white Colour of substrate mycelium orange Colour of diffused pigment — Sugar spectrum ARA + GAL + MAD − XYL − GLU tr RIB + Type A DAP DL Menaquinones (in %) 8/4 − 9/0 (+) 9/2 + 9/4 +++ 9/6 − 9/8 − 16S rDNA homology 96.1% Phospholipids such as not investigated PE, OH-PE, lyso PE, met PE, PC, NPG, PI, PIM, PG, DPG, GL Fatty acids iso 16 +++ iso 15 + iso 17 (+) anteiso 15 (+) anteiso 17 (+) 10-Me 16 − 10-Me 17 + 2-OH 15 + 2-OH 16 + Type 3f MS —

Abbreviations and Explanations for the Identification (+)  1-5% +  5-15% ++ 15-30% +++ >30% DAP diaminopimelic acid ARA arabinose GAL galactose MAD madurose XYL xylose GLU glucose RIB ribose

Sugar types according to Lechevalier et al. 1971 Fatty acid types according to Kroppenstedt 1985 and 1992. 9/4 MK-9 (H₄) 9/6 MK-9 (H₆) 9/8 MK-9 (H₈) MS mycolic acids PE phosphatidylethanolamine OH-PE hydroxy-PE met PE phosphatidimethylethanolamine PC phosphatidylcholine NPG phosphatidylglucosamine PI phosphatidylinositol PIM phosphatidylinositol mannoside PG phosphatidylglycerol DPG diphosphatidylglycerol GL glycolipids Fatty acids iso-16 isohexadecanoic acids or 14-methylpentadecanoic acids 10-Me-18 tuberculostearic acid 2-OH-16 2-hydroxypalmitic acid

Identification of GF270, GF376, GF473, GF578 and GF674 The identification of these strains is based on 5 characteristics which are independent of one another.

1. Morphology and colour of the colonies: short-branched hyphae, which disintegrate into rod- and spore-like elements. The colonies of GF270 and GF376 are salmon-pink (RAL 3022) and those of GF578 and GF674 are light red (RAL 3012).

2. Diamino acids of the peptidoglycan: meso-diaminopimelic acid

3. Mycolic acids: Rhodoccus mycolic acids: The determination of the long-chain mycolic acid was carried out by means of high-temperature gas chromatography. The elution profiles of the mycolic acids of GF270 and GF376 and those of GF473, GF578 and GF674 were identical. The mycolic acid length for GF270 and GF376 was C₃₈ C₄₆ and that for GF473. GF578 and GF674 was C₄₀ C₄₈. The mycolic acid patterns were compared with mycolic acid patterns of Rhodoccus strains. GF270 was identified with a very low correlation factor (0.086) as belonging to Rhodococcus rhodochrous; it was not possible to identify GF376 by this method. The other three isolates GF473, GF578 and GF674 were identified with a very low correlation factor as belonging to Rhodoccus ruber.

4. Fatty acid pattern: unbranched, saturated and unsaturated fatty acids including tuberculostearic acid. The fatty acid pattern is diagnostic of all Rhodococcus genera and closely related Mycobacterium, Nocardia, Dietzia, Tsukamurella and some Corynebacteria species. The identification at the species level was obtained by qualitative and quantitative differences in the fatty acid pattern of GF270, GF376, GF473, GF578 and GF674 with the fatty acid patterns of Rhodoccus species.

5. The 16S rDNA subsequences of GF270 and GF376 were identical (100%), although the comparison of them with the Rhodoccus strains only showed 99.1% similarity to the closest related Rhodococcus rhodochrous. GF473 and GF578 were identical in their 16S rDNA sequence (100%). GF674 differs from GF578 in only one base pair of 500 (99.8%). All three isolates show only a distant relationship with Rhodoccus coprophilus (98.4%). Based on the chemotaxic and molecular biology results, it can be concluded that GF270 and GF376 on the one hand and GF473, GF578 and GF674 on the other hand are strains of 2 new Rhodoccus species. GF270 and GF376 are closely related to Rhodococcus rhodochrous in their 16S rDNA (99.1%), however GF473, GF578 and GF674 are only distantly related to Rhodoccus coprophilus (98.4%).

The enzyme extract can be obtained by professionally customary disruption of the microorganisms, such as, for example, by disruption by means of ultrasound, by means of the French press method or the lysozyme method. This enzyme extract and, of course, also the complete microorganism cells can be immobilized on a suitable support material, customarily embedded in a polymer, for carrying out the process, or absorbed on a suitable support material.

The enzymes according to the invention having nitrile hydratase activity are obtainable from the microorganisms of the genus Amycolatopsis and are able to convert a nitrile into an amide, in particular they are obtainable from Amycolatopsis NA40 (DSMZ 11617).

These enzymes in particular have the following properties:

a) a pH optimum of pH 6.5±1.0

b) a temperature optimum between 35 and 40° C. at a pH of 7.0

c) a K_(M) value for the substrate 3-cyanopyridine of 41.7 mM ±7.7 mM (20 ° C., 45 mM phosphate buffer, pH 7.0) in particular the enzymes have a

d) molecular weight of 106 kDa, such as, for example, determined by SDS-PAGE.

Nitriles can generally be employed as substrates for the biotransformation. Expediently, either aliphatic nitriles having 1 to 10 carbon atoms, optionally substituted by, for example, hydroxyl, amino, halogen or carboxyl, or substituted or unsubstituted aromatic nitriles having 4 to 10 carbon atoms in the aromatic ring system are employed. Aliphatic nitriles having 1 to 10 carbon atoms which can be used are dinitriles, hydroxynitriles, aminonitriles such as, for example, n-octanenitrile, cyanoacetic acid, isocapronitrile, n-valeronitrile, adiponitrile, glutaronitrile, succinonitrile, sebaconitrile, propionitrile, crotononitrile, acrylonitrile, methacrylonitrile, n-butyronitrile or azelanitrile. Aromatic nitriles having 4 to 10 carbon atoms which can be used are nitriles of the general formula

in which R¹ and R² are a hydrogen atom, a halogen atom or C_(1-4-alkyl), F, Cl, Br or I can be used as halogen atom. Methyl, ethyl, propyl, isopropyl, tert-propyl, butyl, isobutyl or tert-butyl can be used as C_(1-4-alkyl). Expedient representatives of the aromatic nitriles of the general formula I or II are 2-, 3- or 4-cyanopyridine, benzonitrile, fluoro-, chloro- or bromobenzonitrile, such as, for example, o-, m- or p-chlorobenzonitrile or 2-chloro-3-cyanopyridine. 3-Cyanopyridine is preferably used as aromatic nitrile having 4 to 10 carbon atoms.

The biotransformation is expediently carried out with addition of substrate in one portion or continuously such that the substrate concentration does not exceed 40% by weight, preferably 30% by weight.

The process is expediently carried out with resting (non-growth) cells.

Suitable media for the biotransformation are those customary in the specialist field, such as, for example, low molecular weight phosphate buffers, HEPES buffers, citrate buffers, borate buffers, the media according to Tables 1 to 3 or modified forms thereof such as, for example, those described in Example 8 (1) or TRIS/HCl buffers.

The biotransformation is expediently carried out at a temperature from 0 to 50° C. and at a pH between pH 4.5 and pH 10, preferably at a temperature from 20 to 40° C. and at a pH between pH 4.5 and pH 10.0.

In a particularly preferred embodiment, the biotransformation can be carried out in the presence of C₁₋₄-alcohols. C₁₋₄-alcohols employed can be methanol, ethanol, propanol or butanol. Methanol is preferably used.

After the reaction, the corresponding amides can then be isolated by customary working-up methods such as, for example, by crystallization.

EXAMPLES Example 1

Growth of Microorganisms of the Genus Actinomadura or Amycolatopsis

a) Various soil samples were inoculated with various nitriles or amides as a C and N source in the enrichment medium according to Table 1 and incubated at 37° C. or 45° C. for 7 10 days. The cultures were then transferred to the same medium and again cultured at 37° C. for 7 10 days. The whole process was repeated 3 times. The cultures were then diluted and plated out in order to obtain individual colonies. The plates were incubated at 37° C. for 5 days. The different colonies were then tested for the desired activity.

Amycolatopsis NA40 (DSMZ 11617) and Amycolatopsis NE31 (DSMZ 11616) were isolated in this way and then grown in the optimized medium (Table 3) for 90 100 h with shaking at 37° C.

Amycolatopsis NA40 (DSMZ 11617) and Amycolatopsis NE31 (DSMZ 11616) Actinomadura spadix E3733 and Actinomadura spadix E3736, azelanitrile served as a C and N source for Amycolatopsis NA40 (DSMZ 11617) and Actinomadura spadix 45A32, n-octanenitrile served as a C and N source for Actinomadura spadix 4501 and cyanoacetic acid served as a C and N source for Actinomadura spadix C15.

b) Amycolatopsis NA40 was cultured in the medium according to Table 3. The culturing was carried out for 2 or 3 to 4 days at a temperature of 37° C. under measured turbidimetrically at 610 nm and the dry weight of the cells was calculated in the following way: weight of the dry cells in mg/ml=OD₆₁₀ nm×0.277. TABLE 1 Enrichment medium Nitrile 2.0 g KH₂PO₄ 7.0 g MgSO₄•7H₂O 0.1 g Vitamin mixture 1.0 ml CoCl₂•6H₂O 2.0 mg FeSO₄•7H₂O 2.0 mg Make up to 1 l with water (pH 6.7-7.3)

TABLE 2 Basal medium Maltose 2.0 g NaNO₃ 1.0 g K₂HPO₄ 0.1 g MgSO₄•7H₂O 0.05 g  Make up to 100 ml with water (pH 7.0)

TABLE 3 Optimized medium D-glucose  4.5 g Meat extract  0.5 g K₂HPO₄  0.1 g MgSO₄•7H₂O 0.05 g CoCl₂•6H₂O  1.0 mg Make up to 100 ml with water (pH 7.0)

Example 2

Biotransformations with Microorganisms of the Genus Actinomadura or Amycolatopsis

(1) For determination of the nitrile hydratase activity, a reaction mixture (2 ml) containing 3-cyanopyridine (1.0 M, 1.0 ml), potassium phosphate buffer (pH 7.0, 0.1 M, 0.5 ml) and 0.5 ml of cell suspension were incubated at 20° C. for 30 min with stirring. The reaction was stopped by addition of 0.2 ml of 3 N HCl. After centrifuging briefly, the nicotinamide formed was determined by means of HPLC (Shimadzu SPD 6A system using a C18 column (Develosil ODS-HG-5. 4.6×250 cm); eluent: 10 mM KH₂PO₄/H₃PO₄ (pH 2.8)/acetonitrile 9:1 (v/v); flow rate: 1 ml/min; the absorption was measured at 230 nm). The specific activity was expressed as .mu.mol of nicotinamide formed/ml/min/OD₆₁₀ nm.

The reaction rates of aliphatic nitriles in the enrichment medium (Table 1) with isolated bacteria is summarized in Table 5, the effects of inducers and cofactors in the basal medium (Table 2) is summarized in Table 4 and the activity comparison of Amycolatopsis to Rhodoccus in the basal medium (Table 2) is summarized in Table 6. The results from Table 4 show that the nitrile hydratase from Amycolatopsis NA40 is constitutively expressed but the cofactor cobalt is necessary for the activity.

(2) Effect of the Temperature on the Growth of NA40

Subcultures (2 ml) were incubated at 37° C. for 2 days in the medium according to Table 3. and then transferred to shaker flasks containing 20 ml of medium according to Table 3. Culturing was carried out at 37, 40, 45, 50 and 55° C. for 3 to 4 days with shaking. The cell growth was measured and the nitrile hydratase activity was determined at 20° C. Table 7 shows the effect of the temperature on the nitrile hydratase activity and on the cell growth. TABLE 4 Effects of inducers and cofactors on the specific activity in the basal medium Total Specific Growth activity activity (OD_(610 nm)) (μmol/ml/min) (μmol/ml/min/OD) Inducer — 1.26 20.9 16.6 ε-Capro- 0.66 9.52 14.5 lactam Crotonamide 3.41 22.9 6.72 Methacrylamide 3.33 2.46 0.74 Butyramide 2.19 0.19 0.88 Propionamide 1.91 0.92 0.48 Urea 1.72 2.97 1.73 Cofactor — 7.97 0.10 0.01 FeSO₄•7H₂O 8.32 3.36 0.40 CoCl₂•6H₂O 8.41 47.8 5.68

TABLE 5 Conversion rates of aliphatic nitriles using the isolated bacteria Growth Total activity Specific activity Strains Substrates (OD_(610 nm)) (μmol/ml/min) (μmol/ml/min/OD) Amycolatopsis NE31 (DSMZ Adiponitrile 2.68 0.377 0.141 11616) Actinomadura E3733 Adiponitrile 1.62 0.347 0.214 spadix Actinomadura E3736 Adiponitrile 1.36 3.00 2.21 spadix Actinomadura 45A32 Azelanitrile 5.81 18.8 3.23 spadix Actinomadura 45O1 n-octane- 7.24 32.2 4.45 spadix nitrile Actinomadura C15 Cyanoacetic 2.04 7.01 3.43 spadix acid Amycolatopsis NA40 (DSMZ Azelanitrile 5.92 33.0 5.57 11617)

TABLE 6 Activity of Amycolatopsis in comparison to Rhodococcus rhodochrous J1 Microorganism Amycolatopsis NA40 Microorganism (DSMZ 11617) Rhodococcus (μmol/ml/min) rhodochrous J1 Activity for  303 314 3-cyanopyridine Purified Purified enzyme enzyme from NA40 from J1 (μmol/min/mg (μmol/min/mg protein) protein) Activity for 1110 371 3-cyanopyridine

(3) For determination of the activity of NA40 with respect to a number of substrates, cells having a dry weight of 0.0388 mg were incubated in the buffer described above. The reaction was started by addition of the appropriate substrate and incubated at 20° C. with shaking for 10 min. The reaction was stopped by addition of 0.2 ml of 2 N HCl and the reaction mixture was briefly centrifuged. The supernatant was analysed by means of HPLC or gas chromatography. Table 8 shows the test conditions for the substrate specificity and Table 9 shows the substrate specificity of resting NA40 cells for various substrates. The respective test conditions are summarized in Table 8 and the results are summarized in Table 9. TABLE 7 Effect of the growth temperature on the nitrile hydratase activity and on the cell growth Total Specific Relative Growth activity activity activity Temperature (mg/ml) (μmol/ml/min) (μmol/ml/min/mg) (%) 37° C. 6.16 4.69 0.761 100 40° C. 5.79 9.89 1.71 225 45° C. 6.56 4.83 0.736 97 50° C. 5.96 1.16 0.195 26

TABLE 8 Test conditions for substrate specificity Substrate Analysis Substrate (mM) method Amide formed 3-Cyanopyridine 1.0 HPLC Nicotinamide 2-Cyanopyridine 0.25 HPLC 2-Picolinamide 4-Cyanopyridine 0.25 HPLC Pyridine- 4-carboxamide Crotononitrile 0.4 HPLC Crotonamide Benzonitrile 0.03 HPLC Benzamide Acrylonitrile 0.4 HPLC Acrylamide o-Chlorobenzonitrile 0.15 HPLC o-Chlorobenzamide m-Chlorobenzonitrile 0.15 HPLC m-Chlorobenzamide p-Chlorobenzonitrile 0.15 HPLC p-Chlorobenzamide 2-Chloro- 0.15 HPLC 2-Chloronicotinamide 3-cyanopyridine Acetonitrile 0.4 GC Acetamide Propionitrile 0.4 GC Propionamide Methacrylonitrile 0.4 GC Methacrylamide n-Butyronitrile 0.4 GC n-Butyramide o-, m-, p-Chlorobenzonitrile and 2-chloro-3-cyanopyridine were added to the reaction mixture dissolved in methanol.

TABLE 9 Substrate specificity of NA40 nitrile hydratase Relative Relative activity activity Substrate (%) Substrate (%) 3-Cyanopyridine 100 m-Chlorobenzonitrile 75 4-Cyanopyridine 168 p-Chlorobenzonitrile 16 2-Cyanopyridine 128 2-Chloro- 126 3-cyanopyridine Benzonitrile 51 Acetonitrile — Crotononitrile 52 Propionitrile 105 Acrylonitrile 115 Methacrylonitrile 130 o-Chlorobenzonitrile 96 n-Butyronitrile 194

(4) Temperature Optimum and Thermal Stability in Resting Cells

The reaction was carried out in the standard reaction mixture for 10 min. The temperature optimum was between 35 and 40° C. (FIG. 5). The cells were then incubated at various temperatures for 30 min and the activity was tested under standard reaction conditions. As can be seen from FIG. 4, the heat stability was 40° C.

(5) pH Optimum and pH Stability in Resting Cells

For this purpose, the reaction was carried out for 10 min in the standard reaction mixture in which the potassium phosphate buffer had been replaced by various 0.1 M buffers. As can be seen from FIG. 6, the pH optimum was between 4.5 and 10. After the cell suspension had been incubated at 20° C. for 30 min at various pHs, the cells were centrifuged. The cells were then washed and resuspended in 0.1 M potassium phosphate buffer pH 7.0. The reaction was carried out for 10 min by addition of 3-cyanopyridine under standard conditions. The enzyme was stable between pH 4.5 and pH 10.0 (FIG. 7).

(6) Accumulation of nicotinamide from 3-cyanopyridine by Means of NA40

The reaction was carried out in a reaction mixture (30 ml), comprising 500 mM 3-cyanopyridine, 40 mM potassium phosphate buffer (pH 7.0) and resting cells (dry weight 2.3 mg). During the reaction, 3-cyanopyridine (500 mM) was added 7 times as soon as it was consumed. In this manner, 4.0 M 3-cyanopyridine was added in the course of 15 h and 3.89 M (475 g/l) nicotinamide was formed, corresponding to a yield of 97.3%. Nicotinic acid was not formed.

Example 3

Identification of Microorganisms of the Genus Amycolatopsis

The following 5 chemotaxonomic markers supported the identification:

1. Diagnostic amino acid of the peptidoglycan: mesodiaminopimelic acid

2. Diagnostic sugars: arabinose and galactose

3. Mycolic acids: mycolic acids absent

4. Menaquinones: MK-9 (H₄)

5. Fatty acid pattern: iso/anteiso-branched and 2-hydroxy fatty acids, small amounts of 10-methyl-branched fatty acids were additionally detected. This fatty acid pattern was found in all representatives of the genus Amycolatopsis (fatty acid pattern 3f)

The combination of these chemical features is diagnostic of all species of the genus Amycolatopsis.

The fatty acid data of the two cultures were compared with the aid of main component analyses using the entries in the fatty acid database. Using this method, it was possible to assign both NE31 and NA40 to the genus Amycolatopsis, an identification of the species, however, was not possible, since the correlation factor was too low. The comparison of the fatty acid patterns of both strains showed, however, that they are two strains of different types.

The result was confirmed by the results of the 16S rDNA sequence analysis.) Here too, assignment to the genus Amycolatopsis took place, but not to any of the Amycolatopsis species described. In this method, the sequence of the 16S rDNA was determined by the direct sequencing of the PCR-amplified 16S rDNA gene. The diagnostic part of the 16S rDNA sequence was compared with the sequences of the type species of the genus Amycolatopsis and related taxa. The result showed that the strain belongs to the genus Amycolatopsis. The highest agreement was found to Amycolatopsis methanolica at 96.9% (NA40) and 96.1% (NE31). Between them, the two isolates showed agreement in the sequences of 99.0%. Our investigations on representatives of the genus Amycolatopsis have shown that for a good species identification the correlation factor must be higher than 99.5%. Since at 96.9% the value is clearly below 99.5%, it can be assumed from this that the two isolates were not representatives of known Amycolatopsis species.

On the basis of the present results, it was not possible to assign the isolates to any of the known Amycolatopsis species. We assume from this that NA40 and NE31 are strains of two new, previously undescribed species of the genus Amycolatopsis.

Identification Characteristics of Microorganisms of the Genus Amycolatopsis Colour of aerial mycelium Colour of substrate mycelium Colour of diffused pigment Sugar spectrum ARA + GAL + MAD − XYL − GLU v RIB + Type A DAP DL Menaquinones (in %) 8/4 − 9/0 (+) 9/2 + 9/4 +++ 9/6 − 9/8 − 16S rDNA homology >99.5% Phospholipids PE + OH-PE + lyso PE − met PE − PC − NPG − PI + PIM v PG + DPG + GL − Type II + OH-PE Fatty acids iso 16 +++ iso 15 + iso 17 (+) anteiso 15 + anteiso 17 (+) 10-Me 16 (+) 10-Me 17 + 2-OH 15 + 2-OH 16 + Type 3f MS — Phospholipids

Example 4

Purification of the Nitrile Hydratase from Microorganism Strain NA40

The strain was cultured at 37° C. for 3 days in the medium according to Table 3. The cells of a 2 l culture were harvested by means of centrifugation and then resuspended in 0.85% strength NaCl solution. The cells were then transferred to 0.1 M potassium phosphate buffer (pH 7.0) comprising 44 mM n-butyric acid and treated with ultrasound. The cell extract was centrifuged and the cell fragments were removed. This extract was used for the enzyme purification.

During the entire purification, potassium phosphate buffer (pH 7.0) comprising 44 mM n-butyric acid was used. As can be seen from Table 10, the enzyme was purified to homogeneity in 3 steps. TABLE 10 Purification of the nitrile hydratase from NA40 Total Total Specific activity protein activity (Units) (mg) (U/mg) Enrichment Cell-free 73,300 1020 71.9 1 extract DEAE- 68,000 110 620 8.62 Sephacel Phenyl- 64,800 61.4 1105 15.4 TOYOPEARL 1 Unit: The amount of enzyme which catalyses the formation of 1 μmol of nicotinamide/min at 20° C.

Example 5

Characterization of the Nitrile Hydratase

(1) Determination of the Molecular Weight, the Subunit Structure and the Cobalt Ion Content

The molecular weight was determined to be 106 kDa by chromatography on a TSK gel column G3000 SW (0.75×60 cm) using a 0.1 M potassium phosphate buffer (pH 7.0) containing 0.2 M KCl and 44 mM n-butyric acid. It was determined that the enzyme consists of 2 different subunits .alpha. and .beta., whose molecular weight was determined to be 30.000 and 26,000 in each case.

FIG. 1 shows the determination of the molecular weight by chromatography on TSK gel G3000 SW.

FIG. 2 shows the determination of the molecular weight by means of SDS-PAGE

FIG. 3 shows the absorption spectrum of the purified enzyme. A broad absorption of 300 400 nm was observed.

(2) Substrate Specificity of the Purified Enzyme

The substrate specificity was determined analogously to Example 2 (1). The results are summarized in Table 11. TABLE 11 Substrate specificity of the purified nitrile hydratase Relative activity (%) Reaction Total with activity Enzyme resting Substrate (M) (μmol/ml/min) reaction cells 3-Cyanopyridine 1.0 17.7 100 100 2-Cyanopyridine 0.25 39.1 221 128 4-Cyanopyridine 0.25 31.6 179 168 Crotononitrile 0.4 11.9 67 52 Benzonitrile 0.03 11.3 64 51 Acrylonitrile 0.4 16.6 94 115 o-Chlorobenzonitrile 0.15 22.4 127 96 m-Chlorobenzonitrile 0.15 15.9 90 75 p-Chlorobenzonitrile 0.15 2.30 13 16 2-Chloro- 0.15 16.0 90 126 3-cyanopyridine Acetonitrile 0.4 — — — Propionitrile 0.4 39.3 222 105 Methacrylonitrile 0.4 22.1 125 130 n-Butyronitrile 0.4 17.9 101 194

1.7 Units of enzyme were added to the reaction mixture (2.0 ml). The reaction mixture contained the respective substrate in 45 mM phosphate buffer (pH 7.0).

(3) Determination of the K_(M) Value

The K_(M) value was determined to be 41.7 mM for 3-cyanopyridine and to be 3.7 mM for acrylonitrile by means of the Lineweaver-Burk diagram. Compared with Rhodococcus rhodochrous J1, which had a K_(M) value relative to 3-cyanopyridine of 200 mM, that of NA40 is significantly lower. This is one of the main advantages of NA40.

(4) Heat Stability and Temperature Optimum

The purified enzyme was incubated for 30 min at pH 7.0 at different temperatures and the conversion of 3-cyanopyridine to nicotinamide was then measured at 20° C. for 1 min. The enzyme was inactivated at a temperature of greater than 40° C. The heat stability was about 40° C. as in resting cells and the temperature optimum as between 35 and 40° C. (FIG. 5).

(5) pH Optimum and pH Stability

For this purpose, the conversion of 3-cyanopyridine to nicotinamide was carried out at 20° C. in a reaction mixture (2.0 ml) comprising various buffers (42.5 mM), 1.71 units of purified enzyme and 500 mM 3-cyanopyridine. The pH optimum was at about pH 6.5±1.0 (FIG. 8).

For determination of the pH stability, 4.2 units of purified enzyme were incubated at 20° C. for 1 h in various buffers (45 mM). A part of the incubated solution, 1.71 units, were added to the standard reaction mixture (cf. Example 2(1)). The remaining activity was determined. The enzyme was stable in a pH range from pH 5 9. The result is shown is FIG. 9.

(6) Inhibitors

The effect of various inhibitors was determined. The results are summarized in Table 12. TABLE 12 Effect of various inhibitors on the purified enzyme Relative Inhibitor mM activity (%) — 100 N-ethylmaleimide 1 97 Iodoacetic acid 1 39 4-Chloromercurobenzoic acid 0.1 69 Sodium azide 1 59 Hydroxylamine 1 37 Phenylhydrazine 1 8 Semicarbazide 1 82 Tiron (disodium salt of 1 110 4,5-dihydroxy-1,3-benzene- disulphonic acid o-Phenanthroline 1 89 α,α′-Dipyridyl 1 100 8-Hydroxyquinoline 1 110 EDTA (ethylenediaminetetra- 1 115 acetic acid Diethyl dithiocarbamate 1 89

Example 6

Effect of Methanol on Resting Cells of NA40

The reaction was carried out for 10 min in the presence of 0 20% (v/v) methanol according to Table 13. As shown in Table 14, the activity is increased by the addition of 5 15% methanol. TABLE 13 Reaction with resting cells Methods {circle around (1)} {circle around (2)} {circle around (2)} {circle around (4)} {circle around (5)} 1.0 M 1.0 ml 1.0 ml 1.0 ml 1.0 ml 1.0 ml 3-cyano- pyridine 0.1 M KPB* 0.9 ml 0.8 ml 0.7 ml 0.6 ml 0.5 ml (pH 7.0) Methanol — 0.1 ml 0.2 ml 0.3 ml 0.4 ml Cell 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml suspension (0.388 mg/ml) Total volume 2.0 ml 2.0 ml 2.0 ml 2.0 ml 2.0 ml *KPB = potassium phosphate buffer

TABLE 14 Effect of methanol on Amycolatopsis NA40 Methanol Relative activity Methods [% (v/v)] [%] {circle around (1)} 0 100 {circle around (2)} 5 123 {circle around (3)} 10 128 {circle around (4)} 15 130 {circle around (5)} 20 105

Example 7

Enrichment of Microorganisms of the Genus Rhodococcus

Various soil samples were inoculated with cyanoacetic acid as a C and N source in the enrichment medium according to Table 1 and the microorganisms Rhodoccus GF270, GF578, GF473 and GF376 were isolated according to Example 1.

Example 8

Biotransformation Using Microorganisms of the Genus Rhodococcus

(1) Heat Stability of the Microorganisms Rhodoccus GF674, Rhodoccus GF578, Rhodoccus GF270 and Rhodoccus GF376 in Comparison with Rhodoccus rhodochrous J1.

For determination of the heat stability, the microorganisms described above were cultured in the following media.

Rhodococcus rhodochrous J1 was cultured for 72 h in the medium described in EP-A 0 307 926. The microorganisms Rhodoccus GF674, GF578, GF270 and GF376 were cultured in the following media at pH 7.0 for up to 96 h:

Rhodoccus GF674 in a medium comprising yeast extract 1.0 g/l, fructose 5.0 g/l, malt extract 10 g/l, acetamide 5.0 g/l, KH₂PO₄ 2.0 g/l, MgSO₄ 7H₂O 0.5 g/l and CoCl₂.6H₂O 10.0 mg. Rhodoccus GF578 in a medium containing yeast extract 1.0 g/l, fructose 15.0 g/l, malt extract 10.0 g/l, acetamide 25.0 g/l, KH₂PO₄ 2.0 g/l, MgSO₄ 7H₂O 0.5 g/l and CoCl₂.6H₂O 5.0 mg. Rhodoccus GF270 in a medium containing yeast extract 12.5 g/l, sodium citrate 5.0 g/l, methacrylamide 7.5 g/l, KH₂PO₄ 2.0 g/l, MgSO₄.7H₂O 0.5 g/l and CoCl₂.6H₂O 30.0 mg. Rhodoccus GF3 76 in a medium containing yeast extract 1.0 g/l, sodium citrate 10.0 g/l, malt extract 15.0 g/l, butyramide 7.5 g/l, KH₂PO₄ 2.0 g/l, MgSO₄.7H₂O 0.5 g/l and CoCl₂.6H₂O 15.0 mg.

The resting cells were then incubated for 15 min at various temperatures and the remaining activity was then determined under the standard reaction conditions according to Example 2(1).

In the course of this it was found that Rhodoccus GF674 had a relative activity of nearly 100% at a temperature of 50° C. and still approximately an activity of 10% at 60° C. Rhodoccus GF578 likewise had 100% relative activity at 50° C. and a relative activity of 20% at 60° C. Rhodoccus GF376 had 100% relative activity up to 50° C., 70% relative activity at 60° C. and nearly still 5% relative activity at 70° C. Rhodoccus GF270 had a relative activity of nearly 100% up to 60° C. and likewise still 5% relative activity at 70° C. In comparison to this, Rhodoccus rhodochrous J1 had 100% relative activity up to 50° C., 80% at 60° C. and no longer any activity at 70° C.

In summary, it can therefore be stressed that Rhodoccus GF270 and GF376 had a better heat stability than J1 and GF270 had the best heat stability.

(2) pH Optimum of the Rhodococcus Strains

The effect of the pH on the nitrile hydratase activity of the Rhodoccus strains GF674, GF578, GF270 and GF376 was determined as described in Example 2(5).

The pH optimum of Rhodoccus GF674 was at pH 7.5 9.5, of GF578 at pH 8 8.5, for GF270 at pH 6 7.0 and for GF376 at pH 6 8.

(3) Substrate Specificity of the Rhodococcus Strains

The substrate specificity is summarized as relative activity in Table 15.

(4) Nicotinamide Accumulation of the Rhodococcus Strains

Analogously to Example 2(6), the Rhodoccus strains GF674, GF578, GF270 and GF376 were cultured with 3-cyanopyridine (about 500 mM). In the course of this Rhodoccus GF674 formed 6 M nicotinamide within 25 h, GF578 5.5 M nicotinamide within 10 h, GF270 about 8.5 M nicotinamide within 20 h and GF376 7.5 M nicotinamide within 20 h.

(5) Tolerance of 3-cyanopyridine on the Activity of the Rhodoccus Strains

In order to test the tolerance of 3-cyanopyridine, resting cells were incubated for 15 min at 20° C. at concentrations of 3-cyanopyridine between 1 and 10% (w/v) and the cells were then removed by centrifugation. After washing the cells with 0.85% NaCl, the remaining activity was measured.

The tolerance of 3-cyanopyridine as a substrate was tested at various substrate concentrations. It was found that at a substrate concentration of 2% (w/v) the nitrile hydratase activity of Rhodoccus rhodochrous J1 decreased by the factor 1.4, the nitrile hydratase activity of Rhodoccus GF674 at a substrate concentration of 4% (w/v) decreased by the factor 1.4, the nitrile hydratase activity of Rhodoccus GF578 remained nearly constant up to a substrate concentration of 8%, the nitrile hydratase activity of Rhodoccus GF270 at a substrate concentration of 4% (w/v) decreased by the factor 1.17 and the nitrile hydratase activity of Rhodoccus GF376 at a substrate concentration of 10% (w/v) decreased by the factor 1.25.

In comparison with the other Rhodoccus strains, Rhodococcus rhodochrous Ji exhibited the poorest tolerance for 3-cyanopyridine. TABLE 15 Rhodococcus rhodochrous Rhodococcus Rhodococcus Rhodococcus Rhodococcus J1 GF674 GF578 GF270 GF376 3-Cyanopyridine 100 (%) 100 (%) 100 (%) 100 (%) 100 (%) 2-Cyanopyridine 45 308 200 15.7 54.3 4-Cyanopyridine 70 231 167 55.6 79.8 Benzonitrile 27 138 85.1 13.4 57.2 2-Chlorobenzonitrile 2.8 64.8 49.0 0 0 3-Chlorobenzonitrile 43 27.8 28.9 8.41 8.63 4-Chlorobenzonitrile 13 0 0 0 0 Acetonitrile 608 1.49 347 659 806 Propionitrile 434 274 132 37.3 245 n-Butyronitrile 352 491 368 181 195 Acrylonitrile 478 147 101 192 257 Crotononitrile 78.2 98.0 124 37.1 110 Methacrylonitrile 86.9 176 122 0 0 

1-11. (canceled)
 12. A process for preparing an amide comprising converting a substrate to an amide by exposing the substrate to an enzyme extract prepared from a microorganism of the species Rhodococcus GF270 as deposited in the Deutschen Sammlung von Mikroorganismen und Zellkulturer GmbHand and having accession number DSM
 12211. 13. A process for preparing an amide comprising converting a substrate to an amide by exposing the substrate to an enzyme extract prepared from a mutant of a microorganism of the species Rhodococcus GF270, as deposited in the Deutschen Sammlung von Mikroorganismen und Zellkulturer GmbHand and having accession number DSM 12211, which is capable of converting a nitrile into an amide.
 14. A process for preparing an amide comprising converting a substrate to an amide by exposing the substrate to an enzyme extract prepared from a microorganism of the species Rhodococcus GF376 as deposited in the Deutschen Sammlung von Mikroorganismen und Zellkulturer GmbHand and having accession number DSM
 12175. 15. A process for preparing an amide comprising converting a substrate to an amide by exposing the substrate to an enzyme extract prepared from a mutant of a microorganism of the species Rhodococcus GF376, as deposited in the Deutschen Sammlung von Mikroorganismen und Zellkulturer GmbHand and having accession number DSM 12175, which is capable of converting a nitrile into an amide.
 16. The process of claim 12, wherein the substrate is an optionally substituted aliphatic nitrile having 1 to 10 carbon atoms.
 17. The process of claim 13, wherein the substrate is an optionally substituted aliphatic nitrile having 1 to 10 carbon atoms.
 18. The process of claim 14, wherein the substrate is an optionally substituted aliphatic nitrile having 1 to 10 carbon atoms.
 19. The process of claim 15, wherein the substrate is an optionally substituted aliphatic nitrile having 1 to 10 carbon atoms.
 20. The process of claim 12, wherein the substrate is an optionally substituted aromatic nitrile having 4 to 10 carbon atoms in the aromatic ring system.
 21. The process of claim 13, wherein the substrate is an optionally substituted aromatic nitrile having 4 to 10 carbon atoms in the aromatic ring system.
 22. The process of claim 14, wherein the substrate is an optionally substituted aromatic nitrile having 4 to 10 carbon atoms in the aromatic ring system.
 23. The process of claim 15, wherein the substrate is an optionally substituted aromatic nitrile having 4 to 10 carbon atoms in the aromatic ring system.
 24. The process of claim 20, wherein the aromatic nitrile is selected from the group consisting of a compound of general formula I:

and a compound of general formula II:

in which R¹ is selected from the group consisting of a hydrogen atom, a halogen atom, and C₁₋₄ alkyl and R² is selected from the group consisting of a hydrogen atom, a halogen atom, and C₁₋₄ alkyl.
 25. The process of claim 21, wherein the aromatic nitrile is selected from the group consisting of a compound of general formula I:

and a compound of general formula II:

in which R¹ is selected from the group consisting of a hydrogen atom, a halogen atom, and C₁₋₄ alkyl and R² is selected from the group consisting of a hydrogen atom, a halogen atom, and C₁₋₄ alkyl.
 26. The process of claim 22, wherein the aromatic nitrile is selected from the group consisting of a compound of general formula I:

and a compound of general formula II:

in which R¹ is selected from the group consisting of a hydrogen atom, a halogen atom, and C₁₋₄ alkyl and R² is selected from the group consisting of a hydrogen atom, a halogen atom, and C₁₋₄ alkyl.
 27. The process of claim 23, wherein the aromatic nitrile is selected from the group consisting of a compound of general formula I:

and a compound of general formula II:

in which R¹ is selected from the group consisting of a hydrogen atom, a halogen atom, and C₁₋₄ alkyl and R² is selected from the group consisting of a hydrogen atom, a halogen atom, and C₁₋₄ alkyl.
 28. The process of claim 24, wherein the reaction is carried out at a temperature from 0 to 50° C. and at a pH of from 4.5 to
 10. 29. The process of claim 25, wherein the reaction is carried out at a temperature from 0 to 50° C. and at a pH of from 4.5 to
 10. 30. The process of claim 26, wherein the reaction is carried out at a temperature from 0 to 50° C. and at a pH of from 4.5 to
 10. 31. The process of claim 27, wherein the reaction is carried out at a temperature from 0 to 50° C. and at a pH of from 4.5 to
 10. 